AUF1 and ALK are found in the same complex. (A) Total proteins (Total Extracts; 5 μg) extracted from NPM-ALK-negative FEPD and -positive Karpas 299, COST, and SU-DHL1 cells were run in parallel with proteins (200 μg) immunoprecipitated with anti-ALK1 (IPαALK) or antiphosphotyrosine (IPαP-Tyr) antibody. The membranes were probed with the anti-AUF1 monoclonal antibody, revealing the presence of different AUF1 isoforms contained in the NPM-ALK complex or linked to phosphotyrosine-containing proteins. IgH and IgL indicate the migration of the heavy and light chains of Ig, respectively. The asterisk indicates a nonspecific protein cross-reacting with the anti-AUF1 monoclonal antibody. (B) Total proteins (Tot. Ext; 5 μg) extracted from NPM-ALK-expressing NIH3T3 cells were run in parallel with proteins (400 μg) immunoprecipitated with anti-ALKc (IPαALK). Control experiments were also performed using NMP-ALK-expressing (“A”) or control cells (“C”) to test for nonspecific AUF1 binding to the IgG1-Sepharose A column (Cont.IP). IgH and asterisk as in panel A. (C) Proteins (400 μg) extracted from NIH3T3 cells stably transfected either with the empty pcDNA3 vector or with different X-ALK fusion cDNAs, as indicated, were immunoprecipitated with monoclonal anti-AUF1 antibody. The presence of ALK protein in the IP was revealed by Western blot analysis using anti-ALK (ALKc) antibody.